The research objective of this project is to investigate the interactions of adeno-associated virus (AAV) with its host cell. The underlying hypothesis is that by understanding these interactions as they apply to the biology of the virus we can contribute to the use of AAV vectors for gene therapy. Staff members focus on two types of interactions: those involved in viral transduction of the target cell, and those between the Rep proteins of the wild type AAV and their cellular partners. Current projects study the tropism and transduction pathways of AAV serotypes, the identification of new serotypes, and the application of vector derived from these viruses to treat diseases by gene therapy. [unreadable] As a result of our work, AAV4 and AAV5 are now accepted as useful vectors for gene transfer and are actively being evaluated in several gene therapy applications including gene transfer to the lung, CNS, eye, and salivary gland. Recent publications describe the use of AAV5 based vectors for the correction of learning defects in MPS VII mice, its use in treating photoreceptor defects that result in blindness associated with achromatopsia, and ability to transduce primary salivary epithelial cells. BAAV vectors are also demonstrating utility in treating hereditary deafness and understanding the underlying biology associated with signal transduction and channel formation in the inner ear . [unreadable] With regard to salivary glands, we have evaluated the ability of salivary glands to function as a depot organ for the expression of blood coagulation factor IX (FIX). FIX deficiency is consider a rare genetic disorder and approximately 80 new cases of severe hemophilia (less than 1% normal circulating levels of FIX) are reported annually. Because of the rare nature of the disease and the great expense of providing purified factor to patients, purified FIX is not provided prophylacticly, but only after severe trauma. However, patients continue to experience spontaneous bleeding episodes, especially in joint and muscle tissue that ultimately lead to joint destruction and severe pain. [unreadable] A recent clinical trial using AAV vectors to direct FIX expression in the liver only demonstrated transient correction, likely as a result of an immune response from the large amount of vector infused into the patient that was needed to transduce the liver. Another possible complication in the trial was related to other viral infections (i.e. HIV, HCV, HBV) that are common in the liver of hemophilia patients, making the liver a difficult target organ in these patients. [unreadable] For several reasons we feel the salivary gland will be an excellent depot organ for this disease and in preliminary studies has demonstrated expression of FIX at therapeutic levels in mice without generating a neutralizing immune response. By optimizing, the promoter, codon sequence, dose of vector, and method of delivery we have achieved expression levels of 30% that of normal human circulating levels hFIX. This would be more than sufficient to treat patients with severe disease. Furthermore we have demonstrated that it is possible to readminister an AAV vector expressing hFIX into mouse salivary glands and achieve the same therapeutic index as the first dose of vector. This is not possible with other depot sites currently being studied. [unreadable] We believe that just as our characterizations of AAV4, AAV5, and BAAV have advanced the field of gene therapy, the development of new vectors also will have an impact on other gene therapy applications, as well as our understanding of parvovirus biology. As a first step in this endeavor, we have developed a PCR based method for rapidly identifying and cloning divergent AAV isolates. We have recently utilized this system to clone a new AAV serotype from a primate adenovirus sample with unique biological and immunological properties. We have termed this isolate AAV12. In contrast to all other reported AAVs, AAV12 cell attachment and transduction does not require cell surface sialic acids or heparan sulfate proteoglycans (HSPG). Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. Feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.[unreadable] Our knowledge of the interactions necessary for cellular transduction with AAV4 and AAV5 clearly has been helpful in defining their biology and likely will identify novel applications for these vectors. Furthermore, the tools we have developed while examining AAV4 and AAV5 tropism can be applied to other novel vectors we have isolated. We expect this information will aid in identifying optimal target cells for the different AAV vectors and also aid in the development of a new generation of vectors for gene transfer with very defined cell tropism. [unreadable] While natural isolates have served as a rich source of vectors for gene transfer, an alternative approach would be to develop vectors with defined tropism. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. Previously we have reported the structure of the AAV5 capsid and more recently we have solved the structure for one of the most antigenically distinct serotypes, AAV4, to 3- resolution.[unreadable] Structural comparison of AAV4 to AAV2 shows conservation of core beta strands and helical secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations, some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes. In the future we plan to use these models to identify receptor binding sites define any structural changes that occur in the viral particles as a result of receptor binding. [unreadable] Sjogrens syndrome is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes. We have demonstrated that delivery of immunomodulatory proteins to the salivary glands of a mouse model of Sjogrens Syndrome can amelorate the gland dysfunction in these animals. In the past we have used female non-obese diabetic (NOD) mouse to model the human disease. While these mice develop exocrine gland infiltrates and, as in SS, a loss of salivary flow that is age and gender dependent, they also develop insulin dependent type 1 diabetes which complicates the study of Sjogrens Syndrome. Furthermore, maintenance of longitudinal stability with respect to onset of the Sjogrens Syndrome phenotype has been reported as problematic. Currently other mouse models of this disease are available and we are reviewing and evaluating them as an alternative to the NOD mouse.